The biosynthesis of early viral mRNA in vaccinia virus (VV)-infected cells is catalyzed by enzymes most of which are not only encoded in the viral genome but also contained within the core of the virus. The availability of these enzymes and their corresponding genes makes the reactions of VV transcription, mRNA processing, and their control, uniquely accessible to study by genetic and enzymological approaches. In this proposal, we have focussed on the VV thymidine kinase (tk) gene, with the aim of understanding the molecular interactions that are involved in its expression and control. We have found that cloned VV genes that are introduced by calcium phosphate coprecipitation into VV-infected cells can be efficiently expressed from within their recombinant plasmids, and we propose to use this system to examine the nucleotide sequences and DNA template structure that are necessary for transcription of the VV tk gene. The functional elements within the tk promoter and the tk mRNA termination/cleavage and polyadenylation sites will be defined by deletion and localized mutagenesis, and the topology of the active templates within the transfected cells will be determined. We will attempt to establish a cell-free system in which purified VV RNA polymerase accurately initiates transcription of the isolated tk gene, and use this system to examine in detail the VV polymerase-promoter interaction. In order to understand the regulation of VV gene expression at the molecular level, we will examine the mechanisms of the transcriptional and translational repressions of the tk gene that occur late in infection. In other studies, the VV tk gene will be expressed from a prokaryotic promoter in tk- E. coli, so that wa may use bacterial genetics to select and characterize nonsense mutants of this gene. Such mutations will be reintroduced into VV to create viable nonsense mutant viruses for the study of nonsense suppression in animal cells. Finally, we will use the tk locus as a selectable site for the insertion and expression of heterologous genes from some cytoplasmically replicating RNA viruses such as poliovirus and respiratory syncytial virus.